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Clostridium difficile-specific antibodies and uses thereof

a technology of clostridium difficile and specific antibodies, which is applied in the field ofclostridium difficile toxinspecific antibodies, can solve the problems of no acute cdad treatment targeting tcda/b, thousands of deaths, and $1 billion in associated costs to the health care system, and achieves reduced affinities of 3 of 4 vhhs, increased thermal stability and gi protease resistance, and increased stability

Inactive Publication Date: 2017-09-26
NAT RES COUNCIL OF CANADA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes a method to improve the stability of small antibodies called VHHs, which are used for treating toxin-related diseases. The method involves adding a disulfide bond to the VHHs, which increases their resistance to digestive proteases and makes them more stable at low pH. This makes the VHHs better suited for oral treatment as they can withstand the harsh environment of the gastrointestinal tract. The disulfide bond mutated VHHs also have improved refolding efficiency and are more effective in neutralizing the toxin. Overall, this method provides a way to enhance the stability and effectiveness of VHHs for treating toxin-related diseases.

Problems solved by technology

Each year in North America, 1-3% of hospitalized patients receiving antibiotics become infected with C. difficile, leading to thousands of deaths and over $1 billion in associated costs to the health-care system (Wilkins and Lyerly, 2003; Kyne et al, 2002; Kelly et al, 1994).
However, there are several other emerging challenges warranting the development of novel therapeutics.
First, there is no acute CDAD treatment targeting TcdA / B.
Second, hypervirulent strains of C. difficile, such as the NAP1 / 027 isolate, over-express TcdA and TcdB (Warny et al, 2005) and have been associated with increased mortality rates and disease severity (O'Connor et al, 2009; Pépin et al, 2005).
The limited success of both oral and systemic anti-toxin immunotherapy in clinical settings has likely been hampered by the high immunoglobulin dose requirements (150-400 mg / kg), the associated costs of these doses, and a lack of published clinical data showing the effectiveness of these treatments.

Method used

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  • <i>Clostridium difficile</i>-specific antibodies and uses thereof
  • <i>Clostridium difficile</i>-specific antibodies and uses thereof
  • <i>Clostridium difficile</i>-specific antibodies and uses thereof

Examples

Experimental program
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Effect test

example 1

Purification of Toxins and Recombinant Fragments

[0133]C. difficile-associated diseases (CDAD) are caused by two high-molecular weight toxins composed of enzymatic, translocation, and cell-receptor binding domains (RBD; FIG. 1A). TcdA and TcdB toxins were purified from natural sources, and recombinant fragments thereof were prepared.

[0134]TcdA and TcdB were isolated from Clostridium difficile strain 10463 (ATCC, Manassas, Va.) as described previously (Keel and Songer, 2007) and were stored in 50 mM Tris-HCl buffer pH 7.5 at 4° C. The SDS-PAGE profile of the purified C. difficile toxins (3 μg per lane; from strain 10463) used is shown in FIG. 1B.

[0135]Recombinant fragments of TcdA (amino acid residues 2304-2710) and TcdB (amino acid residues 2286-2366), which are fragments of the RBD, were cloned (as a BamHI-HindIII fragment for tcdA and a BamHI-EcoRI fragment for tcdB) into pTrcHisB (Invitrogen, Carlsbad, Calif.), transforming E. coli DH5aMCR. Expression was induced by IPTG, cells ha...

example 2

Llama Immunization and Serum Response

[0136]To isolate VHHs which target the RBD of toxin A (TcdA) and toxin B (TcdB), a llama was immunized with recombinant RBD fragments TcdA-RBD-f1 and TcdB-RBD-f1 (FIG. 1A) obtained in Example 1.

[0137]One male llama (Lama glama) was immunized by sub-cutaneous, lower-back injection of TcdA-RBD-f1 and TcdB-RBD-f1 antigens. On Day 1, 200 μg of each antigen diluted in PBS to 1 ml was injected with 1 ml of Freund's Complete Adjuvant (Sigma, St. Louis, Mo.). Three more injections of 100 μg of each antigen+Freund's Incomplete Adjuvant (Sigma) were performed on Days 22, 36, and 50. A final injection of 100 μg of each antigen with no adjuvant was performed on Day 77. Pre-immune blood was drawn before the first injection on Day 1 and served as a negative control. Blood (10-15 ml) was collected on Days 29, 43, 57and 84. Pre-immune and post-immune total serum was analyzed for a specific response to TcdA-RBD-f1 and TcdB-RBD-f1 by ELISA on Day 57 (see below). L...

example 3

Library Construction and Selection of Toxin-binding VHHs

[0139]A hyperimmunized llama VHH library was constructed based on RNA isolated from the serum collected in Example 2.

[0140]Library construction and panning was performed essentially as previously described (Arbabi-Ghahroudi et al, 2009c, 2009b; Tanha et al, 2003). Total RNA was isolated from approximately 5×106 lymphocytes collected on day 84 post-immunization using the QIAAMP RNA Blood Mini Kit (QIAGEN, Mississauga, ON, Canada). About 5 μg of total RNA was used as template for first strand cDNA synthesis with oligo dT primers using the First-Strand cDNA Synthesis Kit (GE Healthcare, Baie-d'Urfé, QC, Canada). The cDNA was amplified by an equimolar mix of three variable region-specific sense primers:

[0141]

MJ1:(SEQ ID NO: 56)5′-GCCCAGCCGGCCATGGCCSMKGTGCAGCTGGTGGAKTCTGGGGGA-3′MJ2:(SEQ ID NO: 57)5′-CAGCCGGCCATGGCCCAGGTAAAGCTGGAGGAGTCTGGGGGA-3′MJ3:(SEQ ID NO: 58)5′-GCCCAGCCGGCCATGGCCCAGGCTCAGGTACAGCTGGTGGAGTCT-3′,

and two antisense C...

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Abstract

The present invention is directed to Clostridium difficile toxin-specific antibodies, compositions, and uses thereof. The anti-toxin antibodies may be specific for either TcdA or TcdB. The invention also includes methods of treating a Clostridium difficile infection, methods of capturing Clostridium difficile toxins, and methods of detecting Clostridium difficile toxins.

Description

CROSS-REFERENCE To RELATED APPLICATIONS[0001]This application is a national phase entry of International Patent Application PCT / CA2011 / 001201 filed Oct. 25, 2011, which claims the benefit of U.S. Provisional Patent Application Ser. No. 61 / 406,254 filed Oct. 25, 2010, the contents of both of which are incorporated herein in their entirety.FIELD OF THE INVENTION[0002]The present invention relates to Clostridium difficile-specific antibodies and uses thereof. More specifically, the present invention relates to Clostridium difficile toxin-specific antibodies and uses thereof.BACKGROUND OF THE INVENTION[0003]Clostridium difficile is a Gram-positive, anaerobic, endospore-forming gastrointestinal pathogen responsible for C. difficile-associated disease (CDAD) in humans and animals with symptoms ranging in severity from mild cases of antibiotic-associated diarrhea to fatal pseudomembranous colitis (Rupnik et al, 2009; Leffler and Lamont, 2009; Songer, 2004; Kelly et al, 1994). Each year in ...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07K16/12G01N33/569
CPCC07K16/1282G01N33/56911C07K2317/22C07K2317/35C07K2317/569C07K2317/76C07K2317/92C07K2317/94G01N2333/33A61P31/04
Inventor HUSSACK, GREGARBABI-GHAHROUDI, MEHDI NATHMACKENZIE, ROGERTANHA, JAMSHID
Owner NAT RES COUNCIL OF CANADA
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